Self-Assembly of the Bio-Surfactant Aescin in Solution : A Small-Angle X-ray Scattering and Fluorescence Study

This work investigates the temperature-dependent micelle formation as well as the micellar structure of the saponin aescin. The critical micelle concentration (cmc) of aescin is determined from the concentration-dependent autofluorescence (AF) of aescin. Values between cmcaescin,AF(10 ◦C) = 0.38 ± 0.09 mM and cmcaescin,AF(50 ◦C) = 0.32 ± 0.13 mM were obtained. The significance of this method is verified by tensiometry measurements. The value determined from this method is within the experimental error identical with values obtained from autofluorescence (cmcaescin,T(WP)(23 ◦C) = 0.33 ± 0.02 mM). The structure of the aescin micelles was investigated by small-angle X-ray scattering (SAXS) at 10 and 40 ◦C. At low temperature, the aescin micelles are rod-like, whereas at high temperature the structure is ellipsoidal. The radii of gyration were determined to ≈31 Å (rods) and ≈21 Å (ellipsoid). The rod-like shape of the aescin micelles at low temperature was confirmed by transmission electron microscopy (TEM). All investigations were performed at a constant pH of 7.4, because the acidic aescin has the ability to lower the pH value in aqueous solution.

Analytical liquid chromatography coupled with mass spectrometry (LC-MS) and determination of ESI-HRMS was performed on an Agilent 6220 TOF-MS (Agilent Technologies, Santa Clara, USA) with a dual ESI-source operating with a spray voltage of 2.5 kV, 1200 HPLC system with autosampler, degasser, binary pump, column oven, diode array detector and a Phenomenex Luna C18 column (3 µm, 100 × 2 mm).Nitrogen was generated by a nitrogen generator NGM 11 and served as nebulizer and dry gas.External calibration was performed with ESI-L Tuning Mix (Agilent Technologies, Santa Clara, USA).

Gradient for preparative RP-HPLC:
Flow: 10 mL/min (Hypersil Gold C18 column, 50 × 21.2 mm, 1.9 µM particles)   Signals belonging to the colour encoded protons can be found in Figure S3 as well as in Table S1.The main difference between both structures in the presence of a tiglic acid residue on C-21 in fraction 1 and an angelic acid residue in fraction 2. The presence of D-xylose instead of D-glucose on the position of the sugar marked with 1''' could not be confirmed.

Determination of pKa-Value of Aescin by Titration
Figure S5: Titration curve of an aqueous aescin solution with a concentration of 0.45 mM.This concentration is near the solubility limit of aescin in pure water.As base a 10 mM sodium hydroxide solution was used.Measurements were performed with a 905 Titrando (Methrom, Filderstadt, Germany).The pKa value of aescin was determined from the buffer region to a value of 4.7 ± 0.2.

1 min 1 minFigure S1 :
Figure S1: UV-intensities (220 nm) of fractions 1 and 2 recorded as function of the retention time tR by analytical liquid chromatography coupled with mass spectrometry (LC-MS).Fractions 1 and 2 were successfully separated.

Figure S2 :Figure
Figure S2: Mass spectra of fractions 1 and 2 recorded by ESI-HRMS (while LC-MS run).Spectra of both fractions show the molecular signal of aescin at m/z = 1331.5(exact mass of aescin = 1330.5g/mol) and the fragmentation patterns looks very similar.This indicates the presence of isomers and the substances contained in each fraction cannot be distinguished only by mass spectrometry.

Figure S4 :
Figure S4: Molecular structures of β-aescin structures present in fractions 1 and 2 obtained by RP-HPLC.Signals belonging to the colour encoded protons can be found in Figure S3 as well as in TableS1.The main difference between both structures in the presence of a tiglic acid residue on C-21 in fraction 1 and an angelic acid residue in fraction 2. The presence of D-xylose instead of D-glucose on the position of the sugar marked with 1''' could not be confirmed.

Figure S6 :
Figure S6: Concentration-normalized autofluorescence of aescin at different concentrations.The fluorescence intensity of aescin was normalized to the aescin concentration in solution.Consequently, the value Iaescin•caescin -1 describes the fluorescence ability of a defined number of aescin molecules.This ability strongly decreases with increasing aescin concentration.Measurements were performed in a buffer solution with constant pH value of 7.4.

Figure S7 :
Figure S7: Surface tension isotherm of aescin in aqueous phosphate buffer at a temperature of 23 °C.Data were recorded with the Du Noüy Ring (DNR) method.The intersection of the two linear regressions defines the cmcaescin,T(DNR) value.The error results from error propagation of the regression parameters.The cmc-value obtained here by the DNR method equals the value obtained from the Wilhelmy plate method.The increase of SFT above cmcaescin,T(DNR) probably results from impurities of the aescin powder[3][4][5].

Figure S8 :
FigureS8: Surface tension (SFT) isotherm of aescin in aqueous phosphate buffer at a temperature of 23 °C (blue area) followed by time dependent measurement of SFT at constant aescin concentration (1 mM, red area).Data were recorded with the Wilhelmy Plate (WP) method.After reaching cmcaescin,T(WP) SFT increases reproducibly with increasing aescin concentration.At constant aescin concentration SFT stays constant over time already after a few minutes.This indicates that the comparably strong increase in SFT after reaching cmcaescin,T(WP) cannot be solely attributed to a nonattainment of an equilibrium state.

Table S1 :
[1,2]R signals of protons in the possible molecular structures of β-aescin shown in FigureS4.The assignment of the signals was done on the basis of works of Yoshikawa et al.[1,2].